Key central metabolites such as ATP, ADP, GTP, GDP, NAD+, NADH and many other small molecules play critical roles in metabolism by binding to, and modulating the activity of protein complexes. This regulation influences cellular energy balance, redox state function, metabolic pathway maintenance and control of upstream and downstream signal transduction pathways in cell growth and differentiation. Metabolic misregulation can result in pathogenic states such as neurodegeneration, cancer and immune disorders. Studies spanning almost a century have led to the identification and biochemical characterization of thousands of enzymes and enzyme isoforms while X-ray crystallographic and NMR spectroscopic analyses have resulted in structural insights into the effects of metabolite binding on protein structure. Our earlier cryo-electron microscopic studies provided detailed insights into the three-dimensional (3D) organization of icosahedral PDH complexes. In similar vein, we have recently extended our work to include cryo-EM analysis of glutamate dehydrogenase, a hexameric inner mitochondrial enzyme that is found in all organisms. Glutamate dehydrogenases catalyze the reversible deamination of L-glutamate to alpha-ketoglutarate, with the production of free NH4+ and transfer of a hydride ion to bound coenzyme NAD+ or NADP+ molecules to yield NADH or NADPH thereby increasing cellular redox potential. The mammalian enzymes localize within the mitochondrion and consist of six 56 kDa monomers arranged as a stacked dimer of trimers. The complex is subject to allosteric regulation by energy-sensor metabolites such as ADP, ATP, GTP, NAD(H), NADP(H) and monocarboxylic acids such as leucine making GDH a cornerstone for integrative metabolic control. Mammalian isoforms of GDH contain in each monomer a catalytic active site domain where glutamate, alpha-ketoglutarate, and adenine nucleotide cofactors bind, a long pivot helix that coordinates closing and opening of the active site cleft in response to bound allosteric modulators, a regulatory cofactor-binding domain, and of a protruding antenna domain which permits sophisticated regulatory control of GDH. Antenna from each trimer associate together and undergo conformational changes during active site cleft widening or closure that have been suggested to mediate inter-subunit cooperativity in response to allosteric regulators. Altered GDH function has been implicated in various metabolic disorders, neurodegenerative diseases, aging and cancer. Over the last two years, we have focused on applying cryo-EM image processing methods to study the conformational landscape and regulation of the 330 kDa bovine GDH ,Glud1, which is highly similar to the human Glud1 isoform, and which can be readily purified. In contrast to our expectation that binding of the inhibitory ligand GTP alone would result in cleft-closure as expected from the crystal structure of the GTP/NADH/glutamate complex, we found that the enzyme displayed an open conformation in the presence of added GTP, essentially indistinguishable from the conformation observed in the presence of the activating ligand ADP. Second, we found that the presence of NADH was also not enough to convert the open-cleft apo form into the closed-cleft form, but instead roughly equal amounts of the open and closed forms were observed, with distinct conformations for NADH. Thus NADH binding is sufficient to partially populate the closed-cleft conformation of GDH, implying that the GTP binding is a secondary event that locks the enzyme in the closed state. Taken together, these findings suggest an elegant mechanism for conformational regulation in GDH, whereby the two alternate conformers of NADH stabilize respectively different conformational states, potentially determining which state is stabilized. An important limitation of the crystallographic studies is that information is restricted to sub-states and complexes that can be crystallized. Thus, while there is an enormous amount of biochemical information on factors that modulate enzyme activity, the structural interpretation is based most of the time on extrapolation from the few crystal structures that are available. A further and potentially profound limitation is that regulation of enzymes frequently involves regions that are conformationally flexible and these regions are either truncated in the constructs used to crystallize the relevant proteins, or are present in conformations constrained by crystal packing forces. Thus, there is a gap in knowledge of how the structures of metabolic enzymes and enzyme complexes change upon binding of single metabolites or of various combinations of the very large number of small molecules they are exposed to in the milieu of the cell. With the advent of near-atomic resolution cryoEM, and automation in data collection and image processing, there is great potential to analyze a large spectrum of conformational states of enzymes including those that may not be amenable to analysis by X-ray crystallography, as we have now shown with cryo-EM studies of glutamate dehydrogenase.

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Borgnia, Mario J; Banerjee, Soojay; Merk, Alan et al. (2016) Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase. Mol Pharmacol 89:645-51
Schauder, David M; Kuybeda, Oleg; Zhang, Jinjin et al. (2013) Glutamate receptor desensitization is mediated by changes in quaternary structure of the ligand binding domain. Proc Natl Acad Sci U S A 110:5921-6
Milne, Jacqueline L S; Borgnia, Mario J; Bartesaghi, Alberto et al. (2013) Cryo-electron microscopy--a primer for the non-microscopist. FEBS J 280:28-45
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Borgnia, Mario J; Subramaniam, Sriram; Milne, Jacqueline L S (2008) Three-dimensional imaging of the highly bent architecture of Bdellovibrio bacteriovorus by using cryo-electron tomography. J Bacteriol 190:2588-96

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